Classical swine fever virus (CSFV), formerly named hog cholera virus, is responsible for classical swine fever (CSF) or hog cholera (HC) (Moennig and Plagemann, 1992. Adv. Virus Res. 41: 53-91; Thiel et al., 1996. eds. Fields, B. N., Knipe, D. M., & Howley, P. M. (Lippincott-Raven, Philadelphia), pp. 1059-1073). Classical swine fever is caused by a small enveloped RNA-Pestvirus within the family Flaviviridae. The natural hosts of the swinel fever virus are solely domesticated and wild swine species.
Pestiviruses are causative agents of economically important diseases of animals in many countries worldwide. Presently known virus isolates have been grouped into four different species: Bovine viral diarrhea virus (BVDV) type 1 (BVDV-1) and type 2 (BVDV-2), CSFV and Border disease virus (BDV) which together form one genus within the family Flaviviridae.
Pestiviruses are small enveloped viruses with a single stranded RNA genome of positive polarity lacking both 5′ cap and 3′ poly(A) sequences. The viral genome codes for a polyprotein of about 4000 amino acids giving rise to final cleavage products by co- and posttranslational processing involving cellular and viral proteases. The viral proteins are arranged in the polyprotein in the order NH2-Npro-C-Erns-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B—COOH (Lindenbach and Rice, 2001. eds. Knipe, D. M., & Howley, P. M. (Lippincott-Raven, Philadelphia), pp. 991-1042). Protein C (=core- or capsidprotein) and the glycoproteins Erns, E1 and E2 represent structural components of the pestivirus virion as demonstrated for CSFV (Thiel et al., 1991. J. Virol. 65: 4705-4712). This also holds true for BVDV. E2 and to a lesser extent Erns were found to be targets for antibody neutralization (Donis et al., 1988. J. Gen. Virol. 69: 77-86; Paton et al., 1992. Virology 190: 763-772; van Rijn et al., 1993 J. Gen. Virol. 74: 2053-2060; Weiland et al., 1990. J. Virology 64:3563-3569; Weiland et al., 1992. J. Virology 66:3677-3682). Erns lacks a typical membrane anchor and is secreted in considerable amounts from the infected cells (Fetzer et al., 2005. J. Virol. 79, 11901-11913; Tews and Meyers 2007. J. Biol. Chem. 282, 32730-32741; Rümenapf et al., 1993. J. Virol. 67, 3288-3295; Magkouras et al., 2008. J. Gen. Virol. 89, 2501-2506); this protein has been reported to exhibit RNase activity (Hulst et al., 1994. Virology 200: 558-565; Schneider et al., 1993. Science 261: 1169-1171; Windisch et al., 1996. J. Virol. 70: 352-358). The function of this enzymatic activity for the viral life cycle is presently unknown. The enzymatic activity depends on the presence of two stretches of amino acids conserved between the pestivirus Erns and different known RNases of plant and fungal origin. Both of these conserved sequences contain a histidine residue (Schneider et al., 1993. Science 261: 1169-1171). Inactivation of the RNase activity residing within the Erns results in an attenuated apathogenic pestivirus which is capable to be used as a modified live vaccine (WO 99/64604).
Vaccines comprising attenuated or killed viruses or viral proteins expressed in heterologous expression systems have been generated for CSFV and BVDV and are presently used. The international patent application WO2005/111201 A1 provides a new generation of a modified live pestivirus vaccine, which comprises a multiple modified pestivirus, having at least one mutation in the coding sequence for glycoprotein Erns and at least another mutation in the coding sequence for Npro, wherein said mutation in the coding sequence for glycoprotein Erns leads to inactivation of RNase activity residing in Erns and/or said mutation in the coding sequence for Npro leads to inactivation of said Npro. In addition, vaccines based on the Chinese viral strain “C” or a derivative thereof has been described (so-called “C-strain vaccines”). It has been shown that four days after application of the vaccine, a complete protection of the animals against virulent CSFV challenge infection can be demonstrated. Further, seven days after vaccination, a complete protection is provided from vertical transmission of challenge virus in carrier animals (de Smit et al., 2001. Vaccine 19: 1467-1476).
However, attempts have been made within the European Union to eradicate CSF through rigorous measures without prophylactic vaccination, which has been forbidden since 1990. Vaccination does represent a legally approved option only as an emergency vaccination in cases when swine fever appears (Art. 19 of the Counsel Directive 2001/89/EC).
Therefore, there is a significant demand for a highly efficient vaccine which allows differentiation between vaccinated and infected animals. Furthermore the vaccine shall exhibit all the advantages of traditional modified live vaccines.
Markers for discrimination between vaccinated and infected animals already have been described. Inter alia the TAV epitope having the amino acids TAVSPTTLR (SEQ ID NO:1) from amino acid position 829 to 837 of the polyprotein (containing Npro-C-Erns-E1-E2-p7-N52-N53-NS4A-NS4B-NS5A-NS5B) and the positions 40 to 48 of the E2 protein, respectively, has been mutated.
WO 2010/074575 A2 for example disclose mutations in the TAV epitope of the E2 protein. The substitution of the proline to asparagine (or two asparagines) has been described as well as mutants having a further asparagine substitution. However, WO 2010/074575 A2 rather concentrates on deletion mutants. Only deletion mutants have been tested in animal experiments.
WO 2011/144360 A1 for example disclose escape variants having an amino acid substitution at amino acid position 830 (alanine to valine), at amino acid position 833 (proline to serine) and at amino acid position 839 (glutamic acid to glycine).
Further, in WO 2007/143442 A2 the CSFV E2 has been mutated to the homologous amino acid sequence of BVDV strain NADL E2 for identifying live attenuated CSFV vaccines.
However, the stability of different substitutions within the TAV epitope in cell culture and after re-isolation of vaccinated animals, so far have not been studied.
In light of this, the problem of the present invention is to provide a stable mutated TAV epitope for generating modified live vaccines which enables discrimination between vaccinated and infected animals.